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Endocrinology Vol. 144, No. 12 5339-5346
Copyright © 2003 by The Endocrine Society

Inhibition of Matrix Metalloproteinases Prevents the Synthesis of Insulin-Like Growth Factor Binding Protein-1 during Decidualization in the Baboon

Zuzana Strakova, Maciej Szmidt, Santha Srisuparp and Asgerally T. Fazleabas

Department of Obstetrics and Gynecology, University of Illinois at Chicago, Chicago, Illinois 60212-7313

Address all correspondence and requests for reprints to: Zuzana Strakova, Ph.D., The University of Illinois at Chicago, Department of Obstetrics and Gynecology, 820 South Wood Street (M/C 808), Chicago, Illinois 60612-7313. E-mail: zstrakov{at}uic.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During pregnancy in the primate, uterine stromal fibroblasts are transformed into decidual cells. Decidualization is associated with extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) play a pivotal role in ECM degradation. We hypothesized that MMPs also contribute to regulation of IGF binding protein-1 (IGFBP-1), a biochemical marker of primate decidual cells. We reported that IL-1ß (10 ng/ml) with steroid hormones [36 nM estradiol-17ß, 1 µM medroxyprogesterone acetate (P), and 100 ng/ml relaxin] induces in vitro IGFBP-1 synthesis. This study demonstrates that IL-1ß also induces stromelysin-1 (MMP-3) mRNA and synthesis of the latent form of MMP-3 (pro-MMP-3) protein in baboon stromal fibroblasts. In contrast, hormones (particularly P) negatively regulate MMP-3 because their addition decreases IL-1ß-induced pro-MMP-3 protein. The ERK and p38 MAPK pathways induced by IL-1ß regulate pro-MMP-3 because inhibitors PD98059 (20 µM) and SB203580 (1 µM) prevent its synthesis. The nuclear factor-{kappa}B inhibitory peptide, SN50 (50 µg/ml), or proteasome inhibitor, MG-132 (1 µM), did not inhibit pro-MMP-3 synthesis but appeared to enhance it. The role of MMPs in IGFBP-1 induction was investigated using a broad-spectrum MMP inhibitor, doxycycline, and specific MMP-3 inhibitor, N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH). Both inhibitors caused the dose-dependent decrease of IGFBP-1. {alpha}-Smooth muscle actin, which is down-regulated during decidualization, was partially up-regulated by doxycycline or N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid. This suggests that {alpha}-smooth muscle actin is modulated by changes in ECM caused by the action of MMPs/MMP-3. Disruption of actin filaments enhances IGFBP-1 induction. Thus, our data imply that IL-1ß-induced MMPs and particularly MMP-3 may up-regulate IGFBP-1 by disrupting the actin cytoskeleton as a result of ECM degradation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UNDERSTANDING THE FACTORS involved in embryo-maternal cross-talk, which result in implantation and the establishment of pregnancy is crucial for reproductive medicine. Decidualization, which involves the transformation of stromal fibroblasts to decidual cells, is the major change that occurs in the primate endometrium after conception. In the human, even in the absence of implantation stromal edema is observed on d 23 of the menstrual cycle and is followed 3–4 d later by a predecidual reaction that begins around the spiral arteries and spreads through the upper two thirds of the endometrium (1). If implantation occurs, the reaction intensifies and forms the decidua of pregnancy. In contrast, the nonhuman primate does not undergo a predecidual reaction during the menstrual cycle (2, 3). However, the stromal fibroblasts undergo extensive modification to form decidual cells after implantation (4), and the conceptus is essential to regulate this process in the baboon (5, 6). In vivo decidualization consists of sequential changes that are characterized by the down-regulation of {alpha}-smooth muscle actin ({alpha}-SMA) expression (7), followed by the induction of cyclooxygenase-2 (COX-2) at the implantation site (8) and the expression of IGF binding protein-1 (IGFBP-1) (5, 6). IGFBP-1 is a major secretory product of the human and baboon decidualized endometrium (9, 10) and is considered to be biochemical marker of decidualization. Decidualization has been the topic of intensive research for years, but the biochemical and molecular events associated with this transition have yet to be fully explained.

IL-1 was identified as one paracrine factor that modulates the communication between human maternal endometrium and embryo (11, 12). We recently reported that IL-1ß in the presence of steroid hormones (36 nM estradiol-17ß, 1 µM medroxyprogesterone acetate, and 100 ng/ml relaxin) induces IGFBP-1 expression in human and baboon stromal fibroblasts (13).

Before implantation, the extracellular matrix (ECM) surrounding the stromal cells is composed of fibrillar collagens (types I and III), collagens V and VI, and fibronectin (14). During decidualization, the ECM is converted to a mixture of residual interstitial proteins and new basal laminar components (14, 15). The matrix metalloproteinases (MMPs) play a pivotal role in ECM degradation (16, 17). Human endometrial stromal cells in culture are known to secrete at least four MMPs: MMP-1, MMP-2, MMP-3, and MMP-9 (18). Each enzyme is released in its latent form. The production of all MMPs except pro-MMP-2 is enhanced in a dose-responsive manner by IL-1 (18). Among them MMP-3 (stromelysin-1) is well suited to initiate an ECM-degrading proteolytic cascade. Its wide range of substrates includes proteoglycans, laminin, nonhelical regions of collagens, pro-MMP-1, pro-MMP-8, pro-MMP-9, and pro-MMP-13 (19, 20).

Dissociation of filamentous actin is essential for IGFBP-1 expression during decidualization (21). Cytoskeleton changes can be also induced from the outside of the cells by disruption of their ECM, which serves as external scaffold of the cells (22). In stromal cells degradation of ECM by MMPs/MMP-3 action can result in intracellular cytoskeleton changes. Thus, the aim of this study was to evaluate the relationship between IL-1ß-induced MMPs/MMP-3 and IGFBP-1 synthesis in stromal fibroblasts during decidualization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human IL-1ß and mouse monoclonal antihuman MMP-3 antibody (catalog no. MAB905) were obtained from R&D Systems, Inc. (Minneapolis, MN). Mouse monoclonal antihuman smooth muscle actin antibody (clone 1A4, catalog no. M0851) was from Dako (Glastrup, Denmark). Mouse monoclonal phospho-ERK 1/2 (Thr202/Tyr204) E10 antibody (catalog no. 9106) and rabbit polyclonal ERK 1/2 (catalog no. 9102), phospho-p38 MAPK (Thr180/Tyr182; catalog no. 9211), and p38 MAPK (catalog no. 9212) antibodies were from Cell Signaling Technology, Inc. (Beverly, MA). Inhibitors PD 98059 (2'- amino-3'-methoxyflavone), SB 203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl) 1H-imidazole], peptide nuclear factor-{kappa}B (NF-{kappa}B) SN50 (peptide sequence: Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro), MG-132 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), [N-Isobutyl-N-(4-methoxyphenylsulfonyl) glycyl hydroxamic acid (NNGH) were from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). Doxycycline hydrochloride was from Sigma Chemical Company (St. Louis, MO). Horseradish peroxidase conjugated secondary antibodies were from Bio-Rad Laboratories (Hercules, CA). An enhanced chemiluminescence kit was from Amersham Life Sciences (Arlington Heights, IL). TriReagent was from Molecular Research Center (Cincinnati, OH). All cell culture supplies were obtained from Invitrogen (Grand Island, NY). Other reagents of cell culture grade were purchased from Fisher Scientific (Itasca, IL), Sigma, or Roche Molecular Biochemicals (Indianapolis, IN).

Isolation of baboon stromal cells
Midsecretory phase (9–12 d post ovulation) endometrial tissue was obtained from adult female baboons (Papio anubis) by endometrectomy or after hysterectomy (n = 12). All animal studies were approved by the Animal Care Committee at the University of Illinois at Chicago. Stromal cells were isolated from baboon endometrial tissues as described in detail previously (23).

Cells were cultured in RPMI 1640 medium supplemented with 0.1 mM sodium pyruvate, 1% penicillin/streptomycin (10,000 U, 10 mg/ml) and 10% heat-inactivated and charcoal-stripped fetal bovine serum (SFBS) at 37 C, 5% CO2. At confluence stromal fibroblast cells were trypsinized, propagated, and used for experiments in first passage. Cell purity was assessed by immunocytochemistry using antibodies against cytokeratin (Dako) and vimentin (Zymed, San Francisco, CA). The purity of the fibroblast cell preparations used in studies was greater than 95%.

RT-PCR detection of MMP-3
Baboon stromal fibroblasts (90% confluent) were placed into culture media with 2% SFBS and treated with IL-1ß (10 ng/ml) for 6 h or 3 d. Cells were lysed with TriReagent (Molecular Research Center) and RNA was extracted using the protocol provided by the manufacturer. Reverse transcription was performed in a final volume of 20 µl with 1 µg total cellular RNA and 50 U Moloney murine leukemia virus reverse transcriptase (Invitrogen) with random hexamers as primers at 42 C for 30 min. PCR amplification was performed with the reverse transcription product, the MMP-3 and H3.3 primers (50 pmol/tube), 2.5 mM MgCl2, 0.5 µl of 5 U/µl Taq polymerase (Invitrogen). After an initial incubation at 94 C for 10 min, 33 amplification cycles consisting of 1 min denaturation at 94 C, 1 min annealing at 60 C, and 2 min extension at 72 C were performed followed by 15 min of final extension at 72 C. Primers specific for MMP-3: 5'-ATTTATTTCTCGTTGCTGCTCATGA-3' and 5'-TATGTTTTGTTCTTTTCCTTATCAG-3' (24) and internal standard H3.3 (23) were added together in a single tube to assure comparable coamplification. The PCR products were analyzed by electrophoresis in 1.5% agarose gels. Size of RT-PCR product for MMP-3 is 573 bp.

In vitro decidualization studies in presence of doxycycline and NNGH
Experiments were performed when the baboon stromal cells reached 80–90% confluence. Culture medium was changed into 2% SFBS. Cells were pretreated with inhibitors (doxycycline, NNGH) at concentrations indicated in figure legends for 1 h before addition of IL-1ß (10 ng/ml) and hormones. The word hormones in this article includes treatment with mix of (final concentration) 36 nM estradiol-17ß, 1 µM medroxyprogesterone acetate (P), and 100 ng/ml highly purified porcine relaxin (kindly provided by Dr. David Sherwood, University of Illinois, Urbana-Champaign). Cell culture medium was changed every 2 d. The medium was collected on d 6 and frozen at -70 C till further analysis. The IGFBP-1 protein present in the culture medium was measured using an ELISA kit (Diagnostic Systems Laboratories, Webster, TX). The cells were rinsed twice with ice-cold PBS and lysed on ice with lysis buffer as previously described (25), and proteins were used for detection of {alpha}-SMA as described bellow.

Analysis of MMP-3 and {alpha}-SMA protein
For MMP-3 protein detection culture media obtained as described above and in figure legends were concentrated (x40) with Microcon centrifugal filter devices YM-10 (Millipore, Billerica, MA). The amounts corresponding to 100–300 µl of original culture media were applied on SDS-PAGE 10% gels. Analysis of protein concentrations in cell lysates with Bradford reagent (Bio-Rad Laboratories) confirmed that there were no significant changes in total cell protein content in cells under the different treatments. For {alpha}-SMA detection, 5 µg of cell lysate protein was used. The separated proteins were transferred into polyvinylidene difluoride (PVDF) membranes. The immunodetection procedures for MMP-3 and {alpha}-SMA antibody followed the protocol provided by the manufacturers. Immunocomplexes were visualized by enhanced chemiluminescence.

Cell signaling studies
Baboon stromal cell were grown to confluence on 60-mm (diameter) dishes and maintained in serum-free medium for 18 h. Specific inhibitors were added at the concentrations indicated in the figure legends 1 h prior the addition of IL-1ß (10 ng/ml). After each of the respective treatments, the cells were rinsed three times with ice-cold PBS (pH 7.4) and lysed on ice with lysis buffer as previously described (25). Protein concentration was determined using the Bradford reagent from Bio-Rad Laboratories. Cell lysate proteins (20–100 µg) were separated by 10% SDS-PAGE under reducing conditions and transferred into PVDF membranes. The immunodetection procedures for each antibody followed the protocols provided by the manufacturers. Immunocomplexes were visualized by enhanced chemiluminescence.

Statistical analyses
One-way ANOVA was used to test the null hypothesis of group differences, followed by a two-tailed t test for pairwise comparisons. Each experiment was repeated three times in triplicate and a P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-1ß induces mRNA expression and protein synthesis of MMP-3 in baboon stromal fibroblasts
To determine whether IL-1ß induces MMP-3 expression in baboon stromal fibroblasts, the RNA isolated from baboon stromal fibroblasts treated with IL-1ß was analyzed by RT-PCR (Fig. 1Go). The mRNA for MMP-3 was detected in samples of baboon stromal fibroblasts treated for 6 h with IL-1ß, and the message was also present after 3 d of treatment.



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  		FIG. 1. IL-1ß induces expression of MMP-3 mRNA in baboon stromal cells. Near confluent baboon stromal cells (d 9 post ovulation) were treated in the absence (control; lanes 1, 2) or presence of IL-1ß (10 ng/ml, lanes 3, 4) for 6 h or 3 d. Total RNA was isolated and subjected to the RT-PCR using specific primers for MMP-3 and the internal standard H3.3 in a single tube. Note that after 6 h and 3 d with IL-1ß treatment, there is significant increase in MMP-3 mRNA expression in baboon cells, compared with controls.

 
The MMP-3 protein (pro-MMP-3) is released from the cells in its latent form. The expected IL-1ß-induced synthesis of pro-MMP-3 protein was confirmed by Western blots in concentrated cell supernatants with antibody specific to MMP-3 (Fig. 2AGo). The size of detected protein (approximately 57 kDa) corresponds to the reported molecular weight for pro-MMP-3.



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  		FIG. 2. Hormones down-regulate MMP-3 protein induced by IL-1ß in baboon stromal fibroblasts. Media from cells treated with IL-1ß (10 ng/ml), hormones (H): 36 nM estradiol-17ß(E), 1 µM medroxyprogesterone acetate (P), and 100 ng/ml relaxin (R) or their combination as indicated in legend were collected on d 6 of treatment. Proteins were concentrated, and amounts equal to 300 µl (A) and 100 µl (B) of original media volumes were separated by 10% SDS-PAGE. After transfer to PVDF membranes, MMP-3 protein was detected by Western blot analysis. Note that presence of hormones down-regulates pro-MMP-3 protein (A and B) and that progesterone appears to be primarily responsible for decrease in pro-MMP-3 protein levels (B).

 
Steroid hormones, particularly progesterone, down-regulate pro-MMP-3 protein synthesis
We previously reported that in vitro decidualization of stromal fibroblasts can occur only in presence of both IL-1ß and hormones (13). IL-1ß alone is not capable of inducing stromal cells to decidualize. The analysis of pro-MMP-3 protein in cell supernatants revealed that IL-1ß-induced pro-MMP-3 protein (Fig. 2AGo, lane 2) was reduced in cells undergoing decidualization (Fig. 2AGo, lane 4). Presence of hormones (Fig. 2AGo, lane 3) alone also decreased pro-MMP-3 levels below the baseline level (control, Fig. 2AGo, lane 1). To further identify which specific hormone used in the hormonal mixture contributes to this down-regulation, we analyzed pro-MMP-3 levels induced by IL-1ß in presence of estradiol-17ß (36 nM), P (MPA, 1 µM), and relaxin (100 ng/ml) individually. The results suggest that down-regulation of pro-MMP-3 is most evident in the presence of P (Fig. 2BGo, lane 5).

Involvement of MAPK pathways in IL-1ß-induced signal transduction in baboon stromal fibroblasts
We previously demonstrated that immediate signal transduction (minutes) after IL-1ß stimulation in primate (baboon and human) stromal fibroblasts includes activation of p38 subfamily of MAPK (13). MAPKs have been identified as universal transducers of different extracellular signals initiated at the cell membrane. MAPKs are family of serine/threonine protein kinases organized hierarchically into three-tiered modules. Three main subgroups of the family have been identified: ERK, Jun-N-terminal kinase, and p38 kinase. The activation of upstream cascade kinases MKK3/6 results in phosphorylation of p38 MAPK at Thr180 and Tyr183 residues. An antibody specific for the phosphorylated form of p38 MAPK detected phosphorylated p38 MAPK (p-p38) presence after IL-1ß treatment (10 and 20 min) in baboon stromal fibroblasts (Fig. 3AGo), confirming our previous results (13). Specificity of p38 activation was verified with the use of SB203580, a specific p38 inhibitor (26). SB203580 (1 µM) significantly inhibited phosphorylation of p38 induced by IL-1ß in 20 min (Fig. 3AGo).



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  		FIG. 3. IL-1ß-induced ERK and p38 phosphorylation is inhibited in the presence of their specific inhibitors. A, Baboon stromal cells were treated with specific p38 inhibitor SB203580 (SB, 1 µM) for 1 h followed by treatment with IL-1ß (10 ng/ml) as indicated in figure legend. The presence of the phosphorylated form of p38 MAPK (p-p38) in cell lysates was detected by Western blot. Membrane was stripped and reprobed with antibody specific to p38 to demonstrate equal protein loading. Note the decrease in p-p38 after SB203580 treatment. B, Baboon stromal cells were treated with IL-1ß (10 ng/ml) for 5–20 min with or without 1 h pretreatment with 20 µM PD98059 (PD) or 1 µM SB203580 (SB) as indicated in the figure. The presence of the phosphorylated form of ERK 1/2 (p-ERK-1 and p-ERK-2) in cell lysates was detected by Western blot with specific antibodies. Membrane was stripped and reprobed with antibody to total ERK-1/2 to demonstrate comparable protein loading. Treatment with 10% fetal bovine serum (FS) was used as positive control. Note IL-1ß induced activation of p-ERK-2 with increasing time of treatment and decrease in p-ERK-2 in presence of PD98059 treatment.

 
IL-1ß induces multiple signal pathways, so we investigated whether in addition to p38 activation; another member of the MAPK subfamily is activated. As detected by Western blot, the best-known member of MAPK subfamily, ERK-2, is also rapidly phosphorylated (5 min) in baboon stromal cells after IL-1ß treatment (Fig. 3BGo). The specificity of activation of ERK-2 by IL-1ß treatment was verified with PD 98059, potent and selective inhibitor of its up-stream MAPK kinase. It selectively blocks the activation of MAPK kinase, thereby preventing phosphorylation and activation of ERK1 and 2 (27). Pretreatment with PD98059 (20 µM) inhibits IL-1ß-induced ERK-2 phosphorylation (Fig. 3BGo). As expected, the p38 specific inhibitor SB203580 (1 µM) does not inhibit phosphorylation of ERK-2 (Fig. 3BGo).

Signal pathways involved in pro-MMP-3 synthesis induced by IL-1ß
Activated ERK and p38 regulate multiple transcription factors important for gene expression. To determine which signal pathways contribute to synthesis of pro-MMP-3 induced by IL-1ß in stromal fibroblasts, we used specific inhibitors of MAPK pathways. Protein analysis of pro-MMP-3 protein secreted during 24 h into cell media of baboon stromal fibroblasts (Fig. 4Go) confirmed inhibition of IL-1ß-induced pro-MMP-3 protein (lane 2) in the presence of the inhibitors PD98059 (20 µM, lane 3) and SB203580 (1 µM, lane 4), suggesting a role of the ERK and p38 pathways in pro-MMP-3 protein regulation. Previously we also reported that IL-1ß induces phosphorylation of inhibitory {kappa}B (I{kappa}B)-{alpha} protein, implying activation of pathway leading to NF-{kappa}B transcription factor in primate stromal fibroblasts (13). To investigate the role of NF-{kappa}B transcription factor in synthesis of pro-MMP-3 induced by IL-1ß in baboon stromal fibroblasts, we used cell-permeable peptide carrying nuclear localization sequence of NF-{kappa}B p50 subunit, which inhibits nuclear translocation of NF-{kappa}B (28). In contrast to previously used inhibitors of ERK and p38 MAPK, the presence of an inhibitor of the NF-{kappa}B pathway (SN50 inhibitory peptide, 50 µg/ml, lane 7) significantly increased amount of pro-MMP-3 protein detected. Similarly, proteasome inhibitor MG-132 (29) (1 µM, lane 5) also significantly increased amount of pro-MMP-3 protein detected. The inhibition of pro-MMP-3 was also observed in the presence of progesterone (1 µM), confirming its specific role for pro-MMP-3 protein down-regulation (Fig. 2BGo, lane 5, Fig. 4Go, lane 6).



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  		FIG. 4. Regulation of pro-MMP-3 protein synthesis. Baboon stromal cells were pretreated with PD98059 (PD, 20 µM), SB203580 (SB, 1 µM), MG-132 (MG, 1 µM), medroxyprogesterone acetate (P, 1 µM), or peptide NF-{kappa}B SN50 (SN, 50 µg/ml) for 1 h followed by IL-1ß (10 ng/ml) treatment for 24 h. The conditioned media were collected and concentrated, and amounts representing 100 µl of original media volume were analyzed for MMP-3 protein by Western blot. The representative blot from three independent experiments is shown. Note that PD98059, SB203580, and P treatment inhibited pro-MMP-3 protein release into supernatants. Treatment with MG-132 and peptide NF-{kappa}B SN50 increased amount of detected pro-MMP-3.

 
Inhibition of MMP-3 protein partially inhibits decidualization
To determine the role of MMP-3 during decidualization, we decided to inhibit its activity both with broad-spectrum MMP inhibitor doxycycline (30) and a specific MMP-3 inhibitor, NNGH (31) (Fig. 5Go). Doxycycline (1–10 µM) was able partially, but statistically significantly (P < 0.05), inhibit decidualization as detected by the decrease in IGFBP-1 synthesis induced by IL-1ß and steroid hormones. The specific MMP-3 inhibitor NNGH (1 µM) had a similar effect on IGFBP-1 synthesis. These results suggest that MMPs in general and perhaps MMP-3 in particular are critical components of a complex series of changes within the cells resulting in the induction of decidualization as measured by the synthesis of IGFBP-1.



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  		FIG. 5. Effect of doxycycline and NNGH on IGFBP-1 protein levels. Baboon stromal cells were pretreated for 1 h with indicated concentrations of broad MMP inhibitor doxycycline (0.1–10 µM) or specific MMP-3 inhibitor NNGH (0.01–1 µM). After 6 d of incubation with IL-1ß in presence of hormones (IL-1ß+H), the IGFBP-1 synthesis was analyzed in collected supernatants by ELISA. The results are plotted as percentage of maximal response (no inhibitor present) to treatment of IL-1ß and H from three independent experiments (actual maximal response values were 393.5 ± 39.6 pg/µg protein). Significant differences (P < 0.05) between drug treatment and maximal response are indicated with an asterisk.

 
Cytoskeleton protein ({alpha}-SMA) during decidualization is affected by MMPs/MMP-3
Because decidualization is a complete morphological and functional cell transformation, it should be accompanied by changes in the cytoskeleton. We previously reported down-regulation of mRNA expression of cytoskeleton protein; {alpha}-SMA during IL-1ß and hormone-induced decidualization (13). Protein levels of {alpha}-SMA were also decreased during this process (Fig. 6Go). It is of interest that the treatment with the broad-spectrum MMP inhibitor, doxycycline (10 µM), or MMP-3-specific inhibitor NNGH (1 µM) partially reversed this effect, suggesting that during decidualization, {alpha}-SMA is modulated by changes in ECM caused by the action of MMPs/MMP-3 (Fig. 6Go). These doses are also similar to those that inhibited IGFBP-1 secretion (Fig. 5Go).



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  		FIG. 6. Effect of doxycycline and NNGH on {alpha}-SMA protein during decidualization. Near-confluent baboon stromal fibroblasts cells were treated with doxycycline (A) or NNGH (B) in indicated concentrations in presence of IL-1ß (10 ng/ml) and hormones (36 nM estradiol-17ß, 1 µM medroxyprogesterone acetate, 100 ng/ml relaxin) for 6 d. Cells were lysed with lysis buffer, and proteins (5 µg) were analyzed for their {alpha}-SMA content by Western blot. The representative blot from three independent experiments is shown. Underneath the blotsis densitometric evaluation of the bands. The staining of the membrane with india ink was used to verify equal protein loading. Note that in comparison with the control, there is a decrease in {alpha}-SMA protein in cells undergoing decidualization induced by IL-1ß in presence of hormones. The presence of doxycycline (10 µM) or NNGH (1 µM) partially reversed this effect as reflected by an increased amount of {alpha}-SMA detected in comparison with IL-1ß and hormones only.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The MMPs have been implicated in the implantation process in a wide range of species (reviewed in Ref. 32). Active remodeling occurs at implantation sites in all mammals and studies in mice have demonstrated that MMP-1 and MMP-3 in particular appear to be involved in tissue remodeling in and around the implantation site (33). Successful implantation after in vitro fertilization has been correlated to high concentrations of both IL-1{alpha} and IL-1ß in the culture media of human embryos (34, 35). Release of IL-1ß by human cytotrophoblasts paralleled their invasive potential; the highest levels were produced by first-trimester cells and the lowest levels by term cells (36). IL-1 induces multiple signal transduction pathways in stromal fibroblasts and leads to the expression of several genes. We previously reported the role of COX-2 induced by IL-1ß in stromal cells during in vitro decidualization (13), and we now focus on the role of MMP-3.

We observed IL-1ß-induced mRNA expression and synthesis of pro-MMP-3 protein in baboon stromal fibroblasts, which is in agreement with previous report in human stromal fibroblasts (18). Pro-MMP-3 is secreted as a 57-kDa single-chain protein. Conversion to MMP-3 involves removal of the NH2-terminal prosequence of approximately 80 amino acids to yield the 45-kDa active enzyme (20).

The pathways induced by IL-1ß leading to synthesis of pro-MMP-3 in primate stromal cells have not been characterized. Our results demonstrate that IL-1ß stimulated the phosphorylation of ERK and p38 MAPKs in a time-dependent manner. Thus, activation of these MAPK pathways is an early event in the IL-1ß-induced signal transduction. The results with specific inhibitors suggest that ERK and p38 pathways are involved in the IL-1ß-induced pro-MMP-3 synthesis. Once activated, MAPKs regulate gene expression via phosphorylation of downstream transcription factors. Thus, we hypothesize that ERK and p38 MAPK could play an important role in the regulation of transcription factors involved in pro-MMP-3 gene expression. Activation of the ERK pathway leads to activation of c-fos. Protein products of c-fos and c-jun are components of a dimeric transcription factor, activator protein-1 (AP-1). A binding site for AP-1 at approximately -90 bp of the promoter sequence is common to MMP-1, MMP-3, and MMP-9 (37, 38).

It is not known how ERK and p38 contribute to MMP-3 expression, but the mechanism could be similar to that recently described in dermal fibroblasts. In dermal fibroblasts two distinct and complementary signaling mechanisms mediating induction of MMP-1 and MMP-3 were identified: AP-1-dependent transcriptional activation via the ERK1/2 pathway and AP-1-independent transcriptional activation via p38{alpha} MAPK by mRNA stabilization (39).

Many of the actions of IL-1ß could be ascribed to its ability to activate the transcription factor NF-{kappa}B (40). This activation is achieved by release of active NF-{kappa}B from an inactive complex with I{kappa}B inhibitory proteins by site-specific phosphorylation of serine residues on I{kappa}B and its translocation to the nucleus (41). Recent work also shows an essential role for the NF-{kappa}B transcription factor in MMP-1, MMP-3, and MMP-9 secretion (42, 43, 44), even though there is not a well-defined NF-{kappa}B binding site in their proximal promoters. In our experiments the NF-{kappa}B SN50 peptide, which binds specifically on NF-{kappa}B p50 nuclear localization sequence and inhibits translocation of the NF-{kappa}B active complex into the nucleus (28), did not prevent pro-MMP-3 induction. In contrast, there was an observed increase in the amount of ILß-induced pro-MMP-3 protein after treatment with NF-{kappa}B SN50. This can suggest that NF-{kappa}B might contribute to expression of some protein that interferes with pro-MMP-3 production or degrades pro-MMP-3. Another possibility is that the MMP-3 gene may be regulated by the cross-talk of AP-1 and NF-{kappa}B in the nucleus (45).

Proteins with regulatory functions are commonly degraded by the 26S proteasome through the ubiquitin-dependent pathway, and it was suggested that inhibition of proteasome leads to accumulation of certain otherwise short-lived proteins. To determine whether pro-MMP-3 synthesis is influenced by proteasome pathway, we used a very potent proteasome inhibitor MG-132 (29). Our data demonstrate that in presence of MG-132, the amount of pro-MMP-3 is increased, suggesting that 26S proteasome plays an important role in the regulation of the pro-MMP-3 protein. Thus, it appears that the steady-state level of pro-MMP-3 is maintained by a precise balance between the rates of its synthesis and its degradation by the proteasome-involved mechanism.

Ovarian steroids play a critical role in the regulation of the reproductive system and also in the regulation of MMP expression in the uterus. Our results in which P specifically mediates the down-regulation of pro-MMP-3 induced by IL-1ß in baboon stromal fibroblasts are similar to observations in isolated human endometrial cells or organ cultures, in which P inhibits expression of MMP-3, MMP-7, and MMP-11 (46, 47).

In our experiments IL-1ß and steroid hormones were added to the baboon stromal fibroblasts at the same time, as would be expected during early pregnancy in vivo. The timing of exposure of stromal cells to IL-1 and progesterone seems to be critical and may impact the progesterone response because prior exposure to progesterone reduces IL-1{alpha} stimulation of MMP-3 or MMP-1 in the human secretory-phase endometrium (48, 49). The action of progesterone is mediated through its receptor (PR), which acts as a ligand-activated transcription factor (50). Besides its effect as an activator of gene transcription, the PR can also influence gene transcription by interplay with other transcription factors, most frequently resulting in an inhibition of gene transcription. It has been reported that the PR can negatively influence gene transcription through direct interaction with the NF-{kappa}B (51, 52) or AP-1 site (53). We would propose that the regulation of MMPs and specifically MMP-3 activity by P is necessary for transformation of stromal cells into fully differentiated decidual cells during pregnancy. Activated MMP-3 is capable of degrading several components of ECM and the basement membrane. Uninhibited MMPs (without presence of conceptus and in absence of hormones) will continue to degrade the ECM, which will result in hemorrhage and tissue sloughing, characteristic of menstruation. On the other hand, the appropriate amount of proteolytic activity of MMPs/ MMP-3 is required for cells to remodel their ECM and differentiate. Thus, one of the mechanisms by which progesterone may mediate decidualization during the implantation process is to regulate IL-1ß-induced MMP-3 proteolysis selectively, thereby allowing decidualization to occur.

It has been demonstrated that disruption of the ECM alters cellular signaling influencing proliferation, death, angiogenesis, differentiation, motility, genomic integrity, and other phenotypes (54). The elaboration of ECM components by fibroblasts not only provides structural support for the surrounding cells but also influences the growth and differentiation programs of these cells. The integral involvement of the MMPs with ECM degradation, cellular invasion, and tissue remodeling suggest a linkage between stromal cell decidualization and MMPs. In this article we provide evidence that MMPs, particularly MMP-3, are involved in the decidualization process because their specific inhibition affects the synthesis of decidualization marker, IGFBP-1. Administration of MMP inhibitor doxycycline during early pregnancy in the rat retards decidual development (55). This correlates with our observation that doxycycline and specific MMP-3 inhibitor NNGH partially inhibited IGFBP-1 produced in baboon stromal cells undergoing decidualization induced by IL-1ß and hormones.

Differentiation represents morphological and functional changes in the cell. Because the cytoskeleton provides necessary intracellular scaffolding to support and organize the cellular components into an appropriate arrangement, its ability to deform and reform is critical for differentiation. The organization of the cytoskeleton is determined primarily by the forces or tension generated within the cytoskeleton by actin and myosin II (56, 57, 58). We previously reported the down-regulation of {alpha}-SMA during in vivo (7) and in vitro (13, 23) decidualization in the primate. In the current article, down-regulation of {alpha}-SMA during decidualization was partially reversed in the presence of a broad-spectrum MMP inhibitor or a specific MMP-3 inhibitor, suggesting their contribution to its regulation. In addition, the inhibition of cytoskeleton down-regulation was also associated with the inhibition of IGFBP-1 synthesis. We propose that MMPs/MMP-3-mediated ECM degradation disrupts the bidirectional signaling between integrins and the cytoskeleton, leading to the down-regulation of {alpha}-SMA. On the basis of results in this study, we propose a hypothetical model for the role of MMP-3 during decidualization (Fig. 7Go). Local MMP-3 induced by conceptus-mediated IL-1ß can regulate, under tight P control, the transformation of stromal fibroblasts to decidual cells by degrading the ECM. This in turn down-regulates {alpha}-SMA and initiates the process leading to the ability of these cells to express IGFBP-1. Inhibition of MMPs prevents the decrease in {alpha}-SMA and inhibits the decidualization process.



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  		FIG. 7. Proposed model of MMP-3 regulation and action in primate decidualization. IL-1ß (presumably coming from conceptus) activates immediate signal pathways including ERK and p38 MAPKs and pathway leading to activation of NF-{kappa}B in stromal cells. ERK and p38 are involved in activation of transcription factors required for MMP-3 expression because their inhibition prevents synthesis of latent form of MMP-3. In contrast, inhibition of NF-{kappa}B translocation or inhibition of proteasome activity increases pro-MMP-3 synthesis induced by IL-1ß. After secretion and activation, MMP-3 acts locally to degrade the ECM surrounding the stromal cells. The presence of steroid hormone P controls the extent of ECM degradation by MMP-3. Degradation of ECM, which serves as cell external scaffold, alters integrin signals transducted into the cell, resulting in cytoskeleton disruption (as reflected by the down-regulation of {alpha}-SMA) and cellular remodeling. Consequently, MMP-induced disruption of cytoskeleton together with the induction of COX-2 (13 ) results in the synthesis of IGFBP-1 in the presence of ovarian steroids. The sites of actions of inhibitors of ERK pathway, PD98059 (PD), p38 pathway, SB 20856 (SB), peptide SN50, which blocks NF-{kappa}B translocation (SN50) and MMP-3 inhibitor (NNGH), are illustrated.

 


   Footnotes
 
This work was supported by NIH Grants HD 39851 (to Z.S.) and HD 36759 (to A.T.F.).

Abbreviations: AP-1, Activator protein-1; COX-2, cyclooxygenase-2; ECM, extracellular matrix; I{kappa}B, inhibitory {kappa}B; IGFBP-1, IGF binding protein-1; MMP, matrix metalloproteinase; NF-{kappa}B, nuclear factor-{kappa}B; NNGH, N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid; P, medroxyprogesterone acetate; PR, progesterone receptor; PVDF, polyvinylidene difluoride; SFBS, charcoal-stripped fetal bovine serum; {alpha}-SMA, {alpha}-smooth muscle actin.

Received April 15, 2003.

Accepted for publication August 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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